Preliminary Study Of Cytotoxic T Lymphocytes (CTLs) By Dendritic Cells(DCs) Pulsed With Exosomes Derived From Glioma Cells

Objective:Designed to conform the glioma primary cells can be isolatedexosomes.We can isolate mononuclear cells from peripheral blood,then Whichcan be converted into dendritic cells through the development eventually.AfterDC are pulsed with exosomes deriverd from glioma cells,they are able tocause the activation effect on cytotoxic T lymphocytes.Methods:Selected a highly malignant glioma specimens from clinicaland made it into a single glioma cell suspension.The suspension was dividedinto two,one of them was used for primary cell culture.We can isolateexosomes secreted by glioma cells using differential centrifugation; Gliomafrozen antigen was prepared by repeated centrifugation and freeze-thawmethod from single cell suspensions. Monocytes can be obtained fromperipheral blood By centrifugation. Under the action of the cytokines IL-4、GM-CSF,These monocytes developed into immature dendritic cells. added thecytokines TNF-αand continued to culture, they becomed mature maturedendritic cells finally; exosomes derived from glioma cells、glioma frozenantigen, respectively,impacted of dendritic cells and then co-cultured with T lymphocytes,We candetect to difference of glioma killing rate in vitro by the MTT.the results werestatistically analyzed in spss16.0.Results:Observerd under the inverted contrast microscope,gliomaprimary cells growed well, adherent cells were fusiform、uniform size、a singleform、showing diffuse distribution. Observered by transmission electionmicroscopy,exosomes were disc-shaped hemisphere and its diamerter wasoabout40~100nm. The hemisphere was surrounded by a lipid bilayermembrane, interior uniformed in different region. monocytes cultured for2~4 hours was the state of the adherent,the monocytes were smaller and shapedsmooth rounds. But2~3days later,the cells gradually became irregular shapefrom the previous round shape. Its volume was larger than ever before andsome small spikes could be found in its surface. Up to the five days,the cellswere clustered and extended many of the larger spikes, their volume wasgreater than ever. A small amount of suspended cells could be observed.Exosomes and glioma frozen antigens were added to dendtritic cells culturemedium.After Co-cultured for7days, the cytokines TNF-αadded to theculture medium and then cultured for24hours. At this point,most of the cellswere in suspension,the surface of the cells stretched out a large number ofdendritic protrusions which were the typical shapes of dendritic cells. WhenE:T=10:1,25:1,50:1, due to the participation of the frozen antigen-DC+Tcells,the killing rate of malignant glioma cells was respectively17.50%±1.13%,21.18%±1.36%,30.20%±1.46%.Due to the participation ofexosome-DC+T cells,the killing rate of malignant glioma cells was32.25%±1.36%,43.04%±1.22%,70.42%±1.40%respectively,two groups could befound they were significantly different(p<0.05).Conciusions:The primary medium of glioma cells could separateexosomes by the method of differential centrifugation,these exosomes hadstrong immune activities. Monocytes and T lymphocytes could be obtainedfrom peripheral blood with lymphocyte lysis and centrifugation. Under theaction of the cytokines IL-4,GM-CSF and TNF-α,these monocytes developedinto mature dendritic cells finally. Dendritic cells were impacted by exosomesderived from glioma cells,which could activate the response of cytotoxic Tlymphocyte,then specifically killed glioma cells.exosomes could cause bettercytotoxic effect than the glioma frozen antigens.